Vol. 36, No. 1
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1992, p. 10-16
0066-4804/92/010010-07$02.00/0 Copyright C 1992, American Society for Microbiology
Potentiation of Antibacterial Activity of Azithromycin and Other Macrolides by Normal Human Serum HENDRIK PRUUL* AND PETER J. McDONALD Department of Microbiology and Infectious Diseases, Flinders Medical Centre, Bedford Park 5042, Australia Received 22 April 1991/Accepted 16 October 1991
The interaction of azithromycin with normal human serum was examined in relation to serum protein binding, MIC, and kinetics of killing of bacteria. While the binding of azithromycin to serum proteins is low (8.5% at a concentration of 0.01 mM in 95% serum), the presence of 40% serum during the MIC test decreased MICs by 26-fold for serum-resistant Escherichia coli and 15-fold for Staphylococcus aureus. Erythromycin had a similar but lesser effect, while roxithromycin was less active against S. aureus in the presence of serum. The rate of killing of E. coli and S. aureus by azithromycin was increased in the presence of serum. The enhancement of antibiotic activity by serum was pH independent, and heat inactivation and preabsorption with homologous bacteria failed to inhibit enhancement by serum. The macromolecular incorporation of [3H]thymidine by E. coli continuously exposed to 2 ,ug of azithromycin per ml (0.25x the MIC) and 40% serum was decreased by 80% at pH 7.8 and by 48% at pH 7.2, while azithromycin alone failed to inhibit incorporation. Inhibition of nucleic acid biosynthesis at pH 7.2 in the presence of serum was also detected with sub-MICs of erythromycin, norfloxacin, and gentamicin but not roxithromycin. A diffusible serum factor was shown to interact with azithromycin to inhibit the growth of E. coli in an agar diffusion assay to detect antibiotic-serum synergy. MATERIALS AND METHODS
It is well recognized that antibiotics which are highly bound to serum proteins have reduced antibacterial activity when they are tested for in vitro activity in the presence of serum proteins, since only free drug is available for antibacterial activity (6). On the other hand, in vitro synergy between antibiotics and serum components, including antibody and complement, has been reported (7, 20, 27) and may also be expressed in the responses of patients during antimicrobial chemotherapy. Standard inhibitory testing of antibiotics is carried out in the absence of host factors, including serum components. Few MIC studies have attempted to include host factors, although it is established that several variables, including serum proteins, phosphates, osmolarity, divalent cations, and pH, can affect the activity of certain antimicrobial agents (32). Azithromycin is a ring-expanded macrolide derivative with a spectrum of activity similar to that of erythromycin, but it has increased in vitro activity against some genera of gram-negative bacteria, including Haemophilus influenzae and Escherichia coli (1, 29). Peak levels of azithromycin in serum do not normally exceed 0.5 ,ug/ml (9). However, levels of azithromycin in tissue are greater than those in serum by a factor of approximately 10-fold (9, 10, 30). In addition, azithromycin has been reported to eradicate Salmonella enteritidis in a mouse model of tissue infection (10). On the basis of the elevated levels of azithromycin in tissue and its in vivo activity against gram-negative bacteria, azithromycin has potentially useful inhibitory activity against a wide range of pathogens, including members of the family Enterobacteriaceae. In this study we determined the serum protein binding of azithromycin, erythromycin, and roxithromycin and compared their inhibitory activities against bacteria in the presence of normal serum under controlled pH conditions. *
Antibiotics. The antibiotics except azithromycin used in this study were obtained from commercial sources; azithromycin was provided by Pfizer PTY, Ltd. Stock solutions were prepared as indicated by the manufacturers and were stored at -20°C. Prior to experiments, the antibiotic solutions were diluted in the appropriate experimental media and used immediately. Radiolabeled ['4C]azithromycin was obtained from Pfizer Inc., Groton, Conn., and [N-methyl'4C]erythromycin was obtained from DuPont, Boston, Mass. Protein binding. The extent of protein binding in pooled normal human serum and purified human serum proteins (Sigma Chemical Co., St. Louis, Mo.) was determined by the ultrafiltration method by using Amicon Centrifree filters with a molecular weight exclusion of 10,000 (Amicon Corp., Lexington, Mass.). Binding was determined in 95% serum and antibiotic concentrations of 0.01 mM. The concentrations of antibiotics in prefiltration solutions and filtrates were determined in triplicate by microbiological assay against Micrococcus luteus ATCC 9341 and Bacillus subtilis ATCC 6633 and by radioassay (Beckman liquid scintillation counter, model LS 3801) of total and filtered radiolabeled antibiotic. Control assays of antibiotics were carried out on unfiltered antibiotic solutions in phosphate-buffered saline. Binding of the antibiotics to the filtration membrane was determined after filtration of the antibiotic in phosphatebuffered saline, and the value was subtracted from that determined in the filtrate. Bacteria and MIC determinations. The E. coli strains used were serum-resistant isolates from infected clinical material. The Staphylococcus aureus strains were the Wood 46 and the ATCC 25923 strains and clinical isolates from infected clinical material. The strains were maintained on blood agar plates and subcultured every 3 weeks. Prior to the experiments, the bacteria were grown overnight in Trypticase soy
Corresponding author. 10
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INTERACTION OF MACROLIDES, SERUM, AND BACTERIA
broth (TSB; Oxoid, Basingstoke, United Kingdom), and the logarithmic phase of growth was obtained by serial dilution in fresh growth medium. MIC determinations were carried out in TSB or Mueller-Hinton broth, which were supplemented with the various serum and serum protein factors at a final concentration of 40%. Human serum albumin (Cohn fraction V) and globulins (Cohn fraction II, III; predominantly gamma and beta proteins) were obtained from Sigma Chemical Co. A modification of the tube dilution method described by Washington and Barry (34) was used. Briefly, immediately prior to bacterial inoculation, the medium was adjusted to pH 7.2, unless otherwise stated, sterilized by filtration, and dispensed in 0.4-ml portions in the MIC dilution tubes. Twofold dilutions of the antibiotics were carried out, and the tubes were inoculated with bacteria to yield 1 x 105 to 5 x 105 CFU/ml. The results were determined visually after 18 h of incubation at 37°C. Sera. Human serum was obtained from eight normal donors and was pooled. The freshly collected serum was divided into 1-ml portions, stored at -70°C, and thawed immediately before the experiment. Serum lacking complement activity was prepared by heating serum at 56°C for 30 min. Serum depleted of specific antibody was prepared by a modification of a previously described method (31). Briefly, freshly thawed serum was incubated with 1010 homologous bacteria for 30 min over ice. The absorption was carried out two times, and bacteria were removed by centrifugation and filtration. Determination of kinetics of killing. The experimental tubes containing 1.8 ml of pH-adjusted TSB with or without 40% serum were inoculated with 0.2 ml of 5 x 107 bacteria and incubated at 37°C for 4 h. The tubes were vigorously mixed (200 oscillations per min) throughout the incubation period. The pH was measured with a pH electrode with an impedance of less than 1,000 MQl (TPS Pty. Ltd., Brisbane, Australia), and the pH was maintained by the addition of 0.1 M NaOH or 0.1 M HCl at 45- to 60-min intervals. Not more than 12% (vol/vol) was added during the pH adjustments. Viable bacteria were determined by sampling 20 ,u1 into sterile saline solution. Portions were plated onto nutrient agar plates and incubated for 18 h at 37°C, and the CFU was counted. CFU was adjusted for the volumes added during
pH adjustment. Determination of [3H[thymidine incorporation. Bacteria incubated in 0.4 ml of pH-adjusted TSB with and without serum and antibiotic. After 100 min, 0.18 MBq (5% [vol/vol]) of [3H]thymidine (specific activity, 1.63 TBq/ mmol; Amersham International Ltd., Amersham, United Kingdom) per ml was added to the experimental tubes, the tubes were incubated for an additional 30 min, and triplicate 5-,ul samples were dispensed into 160 ,u1 of 8% trichloroacetic acid. Precipitated macromolecules were harvested on glass fiber discs by using a PHD cell harvester (Cambridge Technology Inc., Cambridge, Mass.), and the incorporated radioactivity was determined with a Beckman liquid scintillation counter. Determination of synergy between antibiotic and serum by diffusion in agar. Solid bacterial growth medium was prewere
pared by adding 1% (wt/vol) agar (Oxoid) to TSB, and the TSB-agar was liquefied at 121°C for 15 min and was allowed to cool to 55°C. The pH was adjusted to 7.4, and 5 x 106 E. coli per ml of TSB-agar was added. Then, 12.5 ml of the molten inoculated TSB-agar was added to 90-mm-diameter petri dishes, allowed to solidify on a level surface, and stored at 4°C. Wells (diameter, 6 mm) and troughs (30 by 2.5 mm) were cut into the agar, and 25 ,u1 of the test antibiotic was
11
TABLE 1. Protein binding of macrolides antibiotics in serum and effect of serum on MIC MIC (p.g/ml)b at pH:
Bacterium
Antibiotic
Serum binding?
7.2 No serum
E. coli
8 Serum
No
8.5 6.8 0.26 0.7 Azithromycin Erythromycin 39 80 14 13 54 Roxithromycin .95 .160 80
S. aureus Azithromycin Erythromycin Roxithromycin
Serum
serum
0.011 0.33 21
0.56 0.036 0.049 0.006 0.39 0.11 0.06 0.03 0.7 3.7 0.24 1.9
a Antibiotic (0.01 mM) was incubated with rotation for 1 h at 37°C with pooled 95% normal human serum. b Expressed as the geometric mean of at least seven independent determinations.
added to the wells. Diffusion of the antibiotic was allowed to occur for 2 h at 4°C prior to adding the test serum or control buffers (0.1 ml) to the troughs. The plates were incubated for 16 h at 37°C. Enhanced antibacterial activity was indicated by an increase in the zone of inhibition of growth between the antibiotic well and the trough. RESULTS MIC in the presence of serum. The mean MICs of the three macrolide antibiotics tested in TSB at initial pHs of 7.2 and 8 are presented in Table 1. The MICs for S. aureus ATCC 25923 and Wood 46 and the E. coli strain against azithromycin were markedly reduced by increasing the pH of the serum-free growth medium. MICs for S. aureus ATCC 25923 and Wood 46 did not differ, and the MICs for these two strains are combined in Tables 1 and 2. By increasing the pH from 7.2 to 8, the MIC for E. coli was reduced 9-fold and that for S. aureus was reduced 11-fold. Similar decreases in the MICs of erythromycin and roxithromycin occurred when the pH was increased to 8. Serum had a marked effect on the MIC of azithromycin at all pH levels tested. The MIC for E. coli, determined at pH 7.2, decreased from 6.8 to 0.26, a 26-fold reduction, and at pH 8 the decrease in the MIC was 60-fold. The decrease in the MIC of azithromycin for S. aureus in the presence of serum was 15-fold at pH 7.2 and 8-fold at pH 8. The corresponding changes in the MIC in Mueller-Hinton broth at pH 7.2 were 17-fold for E. coli and 40-fold for S. aureus (unpublished data). Serum also lowered the MICs for both E. coli and S. aureus when they were tested with erythromycin in both TSB and Mueller-Hinton broth. However, the MIC of roxithromycin against S. aureus was increased fivefold at pH 7.2 and eightfold at pH 8, while against E. coli, the MICs were decreased by approximately twofold only. Table 1 also includes data on the serum protein binding of azithromycin, erythromycin, and roxithromycin. Azithromycin binding was low and was confined mainly to alpha globulins (unpublished data). Binding of erythromycin was moderate, while .95% of roxithromycin was protein bound. Effect of serum depletion and purified serum proteins on MIC. The MICs of azithromycin and erythromycin were determined in TSB with 40% antibody-depleted serum and serum lacking complement activity, and the results were compared with those obtained with medium containing 40%
12
PRUUL AND McDONALD
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. Effect of serum depletion and purified serum proteins on the MICs of macrolide antibiotics MIC (>tg/ml)' Test medium
E. coli
S. aureus
AZI
ERY
AZI
ERY
TSB
6.8
80
0.56
0.39
40% Serum 40%o Abs-serumb 40% C-def serumc Albumin (70 mg/ml) Globulins (20 mg/ml)
0.26 0.4 0.34 7.5 6.7
6.5 12.5 12.1 .80 .80
0.026 0.038 0.04 0.53 0.67
0.11 0.16 0.14 0.24 0.34
a Expressed as the geometric mean of at least four independent determinations at an initial pH of 7.2. AZI, azithromycin; ERY, erythromycin. b Abs-serum, serum depleted of specified antibody. I C-def serum, serum lacking complement activity.
normal serum (Table 2). The effect of the addition of purified human serum proteins to TSB was also determined. No statistically significant changes (Student's t test) in serumdependent enhanced antibiotic activity against E. coli was detected when complement-inactivated serum or antibodydepleted serum was substituted for normal serum. The addition of 70 mg of albumin (Cohn fraction V) per ml and mixtures of beta and gamma globulins (Cohn fractions II and III) at 20 mg/ml failed to mimic the enhancing effect of serum. Similar results were obtained with S. aureus. pH-controlled kinetics of killing of bacteria by serum and sub-MICs of azithromycin. E. coli was incubated in TSB and 40% serum, and the pH was maintained at pH 7.2 throughout the time course of the experiment. The results are given in Fig. 1. In the presence of 5 ,ug of azithromycin per ml and
40% serum, there was greater than 2-log1o-unit killing at 90 min compared with a 1.25-log1o-unit killing in unsupplemented TSB. Azithromycin was bactericidal against E. coli at sub-MICs between 5 and 1 ,ug/ml only in the presence of serum. The pH of the medium did not vary by greater than 0.25 units during the course of the determinations of viable bacteria under controlled-pH conditions. The effects of various pHs on the bactericidal activity of sub-MICs of azithromycin were determined (Table 3). A variation of the pH of serum-TSB had no effect on the growth of E. coli in the absence of azithromycin, except at pH 7.8. At all concentrations of azithromycin in the absence of added serum, there was an increase in bactericidal activity with increasing pH. The addition of 40% serum enhanced the bactericidal activity of azithromycin at all pH levels tested except at elevated pH in the presence of 1 ,ug or greater of azithromycin per ml. Under these conditions, azithromycin alone had significant bactericidal activity. The rate of killing of S. aureus by azithromycin at pH 7.2 was slower than that of E. coli (Fig. 2). Even at lOx the MIC, there was less than 1-log1o-unit killing of S. aureus, compared with a 1.65-log1o-unit killing of E. coli within 2 h at 1 x the MIC. However, the effect of serum on the activity of azithromycin was similar. At lOx the MIC, azithromycin was bactericidal against S. aureus in both the presence and absence of serum and there was approximately a 0.7-log1ounit increased killing after 2 h of incubation in the presence of serum. At sub-MICs (.0.25 ,ug/ml), azithromycin retained antibacterial activity in the presence of serum, indicating a potentiation of azithromycin activity by serum. In the absence of serum, bacterial growth occurred at azithromycin concentrations of less than 0.5 ,ug/ml. Effect of serum on [3H]thymidine incorporation. The effect of sub-MICs of azithromycin on the ability of E. coli to
LL
0 0
0
0-
-1i ~ 0
I
1
I
iIIII
1 4 0 2 3 4 Time (hours) FIG. 1. Kinetics of killing of E. coli by azithromycin in the presence of 40% normal human serum at pH 7.2. Each datum point indicates the mean and 1 standard deviation. (A) 5 ,u.g of azithromycin per ml; (B) 2 ,ug of azithromycin per ml; (C) 1 ,ug of azithromycin per ml; (D) 0.5 ,g of azithromycin per ml. 0, 40% serum; 0, no serum; A, no antibiotic, 40% serum. 2
3
INTERACTION OF MACROLIDES, SERUM, AND BACTERIA
VOL. 36, 1992
13
TABLE 3. Effect of pH on the bactericidal activity of azithromycin against E. coli in the presence of 40% serum log1o change in CFU at 2 h at pH':
Medium
7.2
7.4
7.6
7.8
2 ,ug of AZIb per ml 2 p.g of AZI per ml + serum
0.13 (0.36)" -1.92 (0.3)
-1.26 (0.41) -2.07 (0.14)
-2.15 (0.46) -2.4 (0.23)
-2.5 (0.5) -1.75 (0.24)
1 p.g of AZI per ml 1 ,ug of AZI per ml + serum
1.11 (0.18) -0.82 (0.38)
0.03 (0.19) -1.14 (0.2)
-0.23 (0.46) -2.27 (0.45)
-1.64 (0.17) -1.4 (0.4)
0.5 ,ug of AZI per ml 0.5 ,ug of AZI per ml + serum
1.85 (0.4) 0.75 (0.19)
1 (0.25) 0.02 (0.3)
0.87 (0.09) 0.01 (0.29)
0.13 (0.15) -1.3 (0.2)
Serum alone
1.4 (0.18)
1.46 (0.25)
1.42 (0.41)
0.72 (0.14)
a Expressed as the geometric mean (standard error of the mean) of at least three independent determinations. b AZI, azithromycin.
incorporate [3H]thymidine into macromolecular nucleic acid is shown in Fig. 3. Bacteria were incubated with various concentrations of azithromycin in the presence and absence of serum for 100 min before the addition of [3H]thymidine, and cells were harvested at 130 min. Under the conditions of these experiments, sub-MICs of azithromycin in the absence of serum failed to inhibit incorporation. At all concentrations of antibiotic tested in the presence of serum, inhibition of incorporation of thymidine was greater at pH 7.8 than it was at pH 7.2. To achieve 50% inhibition at pH 7.8, a concentration of 0.8 ,ug of azithromycin per ml was required, while at pH 7.2, 2 ,ug of azithromycin per ml was necessary to achieve the same degree of inhibition. Several antibiotics were compared for their ability to inhibit thymidine incorporation in the presence of serum (Table 4). All tested antibiotics except roxithromycin inhib-
ited thymidine uptake at pH 7.2. Norfloxacin at 0.25 x the MIC was the most effective, while ampicillin and chloramphenicol were not as effective as the two active macrolides azithromycin and erythromycin. An increase in the pH to 7.8 increased inhibition by the macrolides and gentamicin, but not by norfloxacin and ampicillin. Detection of the potentiation of azithromycin activity by serum in an agar diffusion assay. Diffusion of azithromycin in TSB-agar inhibited bacterial growth in clearly defined circular zones of inhibition, as demonstrated in Fig. 4B. Similar inhibition zones were observed with phosphate-buffered saline at pH values of 7.2 to 8. The patterns of inhibition differed when the troughs were loaded with normal serum (Fig. 4A). Elliptical zones were observed, with bacterial growth inhibition extending to a greater distance from the center of antibiotic diffusion when compared with the inhi-
LL
I
C
()
0
4 3 4 0 12 Time (hours) FIG. 2. Kinetics of killing of S. aureus by azithromycin in the presence of 40% normal human serum at pH 7.2. (A) 5 ,ug of azithromycin per ml; (B) 0.5 ,ug of azithromycin per ml; (C) 0.25 ,ug of azithromycin per ml; (D) 0.1 ,ug of azithromycin per ml. *, 40% serum; 0, no serum; A, no antibiotic, 40% serum. 0
1
2
3
PRUUL AND McDONALD
14
ANTIMICROB. AGENTS CHEMOTHER.
E 2 CD .0
50-
1000
1
2 3 Azithromycin (pg/ml)
4
5
FIG. 3. Inhibition of [3H]thymidine incorporation by E. coli in the presence of serum and sub-MICs of azithromycin. 0, pH 7.8; 0, pH 7.2. Each point represents the mean ± standard deviation of at least three independent determinations. [3H]thymidine uptake is expressed as percent uptake in the absence of serum.
bition zones with troughs loaded with buffer. Antibiotic potentiation by serum was critically dependent on the distance of diffusion between serum and antibiotic, and at a concentration of 75 ,ug of antibiotic per ml at pH 7.2, enhanced antibacterial activity was observed at diffusion distances of between 3 and 5 mm. Potentiation between erythromycin and roxithromycin with serum was unable to be determined because these antibiotics failed to inhibit the growth of E. coli in agar at suitable concentrations of these antibiotics. DISCUSSION The low serum protein binding of azithromycin shown in Table 1 is consistent with the previously described concentration-dependent binding properties of the drug (9). At a molarity equivalent to that of azithromycin, erythromycin binding is approximately fivefold higher than that of azithromycin. At these comparatively low levels of protein binding, an increase in the MIC in the presence of serum proteins is TABLE 4. Effect of serum on [3H]thymidine uptake by E. coli in the presence of sub-MICs of antibiotics Antibiotic
X
Azithromycin Erythromycin Roxithromycin Ampicillin Norfloxacin Gentamicin Chloramphenicol a Expressed as
MICb
0.25 0.25 0.25 0.5 0.25 0.25 0.25
%
[3H]thymidine uptake"
pH 7.2
pH 7.8
52.5 (8)c 55.9 (18) >100 76.3 (17) 10.5 (8) 58.2 (5) 81.1 (14)
20.3 (4) 25.5 (3) >100 75.5 (15) 11.7 (3) 18 (10) 67.1 (9)
ihe percent of uptake by E.
coli in the absence of 40%
serum. b
Determined in the absence of serum at pH 7.2. Numbers in parentheses indicate the standard error of the means of at least three independent determinations. c
FIG. 4. Synergy between azithromycin and serum against E. coli in TSB-agar. (A) Normal serum (trough). Upper wells, 75 p.g of azithromycin per ml; lower wells, (left to right) 40, 50, 75, and 100 p,g of azithromycin per ml. (B) Phosphate-buffered saline; pH 8 (trough). Azithromycin was present at the concentrations described above for panel A.
not expected (25). In contrast, roxithromycin is highly bound by whole serum, and the presence of serum in the MIC test increased the MIC against S. aureus fivefold at pH 7.2. Increased MICs in serum-supplemented broth have been reported previously (16a) for roxithromycin against grampositive organisms and for other highly protein bound antibiotics (5, 16, 24, 32). However, roxithromycin decreases the MIC for E. coli by two- to fourfold in serum (Table 1). The reason for this is not clear, but is is probably not due to complement since heat-inactivated serum retains synergistic activity (Table 2). Perhaps the affinity of roxithromycin for binding sites of E. coli is greater than that for serum proteins, resulting in a concentration gradient that reverses the binding to serum proteins. The inhibitory effect of protein binding of antibiotics is normally detected in the MIC test by comparing the MICs in broth and serum. Anomalous enhancement activity of serum has been reported (13, 18, 19, 28, 33). Serum enhancement of antibiotic activity can be attributed to various factors, including the influence of serum on growth of the bacteria (2), serum alpha-2-globulin (22), changes in the pH of the test media (8), antibacterial peptides (4), or as yet unidentified synergistic host factors (18, 19, 21). Leggett and Craig (19) have shown that the increases in the MICs of ceftriaxone and other highly protein bound cephalosporins observed in the presence of albumin were significantly less than those predicted when they were determined in serum-containing medium and were not predicted from the protein-binding properties of the antibiotics. This serum enhancement of cephalosporin activity was limited to gram-negative species. However, the serum enhancement with macrolides de-
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INTERACTION OF MACROLIDES, SERUM, AND BACTERIA
scribed here occurs with staphylococci as well as E. coli. Other body fluids have been shown to enhance antibacterial activity. In a study determining the activity of antimicrobial drugs in duodenal-pancreatic secretions, Mett et al. (21) demonstrated enhanced bactericidal activity of clioquinol, chloramphenicol, and trimethoprim against E. coli and S. aureus, while other antibiotics, including colistin and tetracycline, failed to do so. This activity was not dependent on pH or iron and was heat stable. Macrolide antibacterial activity is sensitive to the pH of the test medium, and the data in Table 1 confirm the previous findings of Barry et al. (1), and in addition, the data demonstrate that. MICs are reduced in the presence of serum at elevated pHs as well as at physiological pH levels. In this study the pHs of the MIC tubes were controlled by adjusting the pH prior to incubation. However, the pHs of serum preparations are known to be unstable (3). In addition, bacterial metabolites may also influence the pH of the test medium. These factors make the continuous control of pH in MIC tubes difficult. In order to control pH more closely and to determine the early kinetics of antibacterial activity, bacteria were exposed to the combined activity of serum and azithromycin under conditions in which the pH was monitored and adjusted in the first 4 h of interactions. The experiments demonstrate that at constant pH, the presence of serum enhances the extent and early rate of killing of bacteria. The bactericidal activity of azithromycin against E. coli develops rapidly, while that against S. aureus is slower to develop (Fig. 1 and 2, respectively). This is in contrast to the findings of Retsema et al. (29) and may be due to differences in the pH of the medium and the effect of bacterial metabolites on pH. Figure 2 demonstrates that the growth of S. aureus at a controlled pH of 7.2 remains suppressed at 0.1 ,ug of azithromycin per ml (0.2 x MIC), and at levels near the MIC, the antibiotic is bactericidal in the presence of serum. The achievable in vivo level of azithromycin in serum is approximately 0.5 ,ug/ml (9), and significant antistaphylococcal activity at this concentration can be expected in the presence of serum. Inhibition of E. coli at pH 7.2 requires 10 times increased levels of azithromycin; these concentrations are achievable in various tissues (9, 10) but not in serum. The combination of serum and azithromycin has powerful antimetabolic activity. At sub-MICs of antibiotic, incorporation of thymidine into macromolecular nucleic acid is dramatically inhibited in the presence of serum (Fig. 3). The effect was enhanced at an elevated pH because of the intrinsic increased activity of azithromycin at high pH. At physiological pH, approximately one-third of the MIC of azithromycin was required to inhibit nucleic acid metabolism by 50%. These results indicate that extensive metabolic disruption occurs under conditions which increase the bactericidal activity of the antibiotic. In addition, exposure of bacteria to antibiotic has been shown to alter bacterial surface structures that are not directly related to the site of action of the antibiotic (14, 15, 17) and that modify subsequent interactions with host factors, including serum components and neutrophils (26). Several other antibiotics tested also showed synergistic antimetabolic activity in the presence of serum, including erythromycin, gentamicin, and ampicillin, but not roxithromycin (Table 4). Only the two active macrolides and gentamicin demonstrated increased inhibitory activity at elevated pH, and this may have been due to their increased antibacterial activity under those conditions. The inhibitory effect in the presence of erythromycin required 20 ,ug of the antibiotic per ml, a level that is
15
not achievable in vivo, while azithromycin was active at levels that are achievable in tissue and serum. The MICs of azithromycin for most E. coli strains, as determined in conventional broth media, are greater than those that are achievable in the blood, suggesting that it would be ineffective in the eradication of these bacteria. However, the eradication of S. enteritidis by azithromycin, but not by cefaclor, which was shown to have similar in vitro potency, has been demonstrated in a mouse tissue model of liver and spleen infection (10). The positive response was attributed to the high levels achieved in tissue and the long half-life in tissue. In addition, potentiation of antibiotic activity by serum and a rapid onset of bactericidal activity may also contribute to the efficacy of azithromycin against gram-negative bacteria. The serum factor(s) responsible for the potentiation of macrolide activity and its mode of action are not known. Changes in the bacterial growth phase and the composition of bacterial surfaces brought about by serum factors may modify bacterial membrane permeability, alter antibiotic accumulation kinetics, or enhance antibiotic binding to active sites. Wild-type gram-negative bacteria with intact outer cell wall structures exclude hydrophobic agents (23). Since macrolides are hydrophobic (11), gram-negative bacteria are normally resistant to this class of antibiotics. Disruption of the hydrophobic barrier of gram-negative bacteria by permeabilizers (12) has been shown to increase the susceptibilities of these bacteria to antibacterial agents that are normally excluded by the intact outer cell wall. For example, wildtype gram-negative bacteria that are relatively resistant to polymyxin are sensitized after chelation treatment of the bacteria with EDTA-Tris (27). The increased susceptibilities of rough, cell wall-deficient bacteria compared with those of smooth strains to erythromycin and other hydrophobic antibiotics have been reviewed (11). We detected early inhibition of nucleic acid biosynthesis in the presence of sub-MICs of some antibiotics and serum, indicating increased intracellular permeation and activity of azithromycin and other antibiotics in the presence of serum factors. We postulate that normal human serum interacts with a range of bacteria to alter their susceptibilities to azithromycin and other macrolide antibiotics with low protein binding characteristics. While high levels of serum protein binding interferes with serum synergy against staphylococci, it may be retained against E. coli. ACKNOWLEDGMENT This work was supported by Pfizer Central Research Division, Pfizer Inc. REFERENCES 1. Barry, A. L., R. N. Jones, and C. Thornsberry. 1988. In-vitro activities of azithromycin (CP 62993), clarithromycin (A-56268; TE-031), erythromycin, roxythromycin, and clindamycin. Antimicrob. Agents Chemother. 32:752-754. 2. Brown, R. W., and P. Williams. 1985. Influence of substrate limitation and growth phase on sensitivity to antimicrobial agents. J. Antimicrob. Chemother. 15(Suppl. A):7-14. 3. Bryan, C. S., S. R. Marney, Jr., R. H. Afford, and R. E. Bryant. 1975. Gram-negative bacillary endocarditis: interpretation of the serum bactericidal test. Am. J. Med. 58:209-215. 4. Carroll, S. F., and R. J. Martinez. 1981. Antibacterial peptide from normal rabbit serum. Biochemistry 20:5973-5981. 5. Craig, W. A., and C. M. Kunin. 1976. Significance of serum protein and tissue binding of antimicrobial agents. Annu. Rev. Med. 27:287-300. 6. Craig, W. A., and P. G. Welling. 1977. Protein binding of
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PRUUL AND McDONALD
antimicrobials: clinical pharmacokinetics and therapeutic implications. Clin. Pharmacokinet. 2:252-268. 7. Dutcher, B. S., A. M. Reynard, M. E. Beck, and R. K. Cunningham. 1978. Potentiation of antibiotic activity by normal serum. Antimicrob. Agents Chemother. 13:820-826. 8. Eagle, H., M. Levy, and R. Fleischman. 1952. The effect of pH of the medium on the antibacterial action of penicillin, streptomycin, chloramphenicol, terramycin, and bacitracin. Antibiot. Chemother. 11:563-575. 9. Foulds, G., R. M. Shepard, and R. B. Johnson. 1990. The pharmacokinetics of azithromycin in human serum and tissues. J. Antimicrob. Chemother. 25(Suppl. A):73-82. 10. Girard, A. E., D. Girard, A. R. English, T. D. Gootz, C. R. Cimochowski, J. A. Faiella, S. L. Haskell, and J. A. Retsema. 1987. Pharmacokinetics and in vivo studies with azithromycin (CP-62,993), a new macrolide with an extended half-life and excellent tissue distribution. Antimicrob. Agents Chemother. 31:1948-1954. 11. Hancock, R. E. W., and A. Bell. 1988. Antibiotic uptake into gram-negative bacteria. Eur. J. Microbiol. Infect. Dis. 7:713-720. 12. Hancock, R. E. W., and P. G. W. Wong. 1984. Compounds which increase the permeability of the Pseudomonas aeruginosa outer membrane. Antimicrob. Agents Chemother. 26:48-52. 13. Hardy, D. J., R. N. Swanson, R. A. Rode, K. Marsh, N. L. Shipkowitz, and J. J. Clement. 1990. Enhancement of the in vitro and in vivo activities of clarithromycin against Haemophilus influenzae by 14-hydroxy-clarithromycin, its major metabolite in humans. Antimicrob. Agents Chemother. 34:1407-1413. 14. Hirashima, A., G. Childs, and M. Inouye. 1973. Differential inhibitory effects of antibiotics on the biosynthesis of envelope proteins of Escherichia coli. J. Mol. Biol. 79:373-389. 15. Iida, K., and M. Koike. 1974. Cell-wall alterations of gramnegative bacteria by aminoglycoside antibiotics. Antimicrob. Agents Chemother. 5:95-97. 16. Jones, R. N., and A. L. Barry. 1987. Antimicrobial activity of ceftriaxone, cefotaxime, desacetylcefotaxime, and cefotaximedesacetylcefotaxime in the presence of human serum. Antimicrob. Agents Chemother. 31:818-820. 16a.Jones, R. N., and A. L. Barry. 1987. Unpredictable influence of human serum on antimicrobial activity of erythromycin and three oxime ether macrolides. Eur. J. Clin. Microbiol. 6:81-82. 17. Klainer, A. S., and R. R. B. Russell. 1974. Effect of the inhibition of protein synthesis on the Escherichia coli cell wall envelope. Antimicrob. Agents Chemother. 6:216-224. 18. Lang, S. D. R., G. L. Cameron, and P. R. Mullins. 1981. Anomalous effect of serum on the antimicrobial activity of cefoperazone. Drugs 22(Suppl. 1):52-59. 19. Leggett, J. E., and W. A. Craig. 1989. Enhancing effect of serum ultrafiltrates on the activity of cephalosporins against gramnegative bacteria. Antimicrob. Agents Chemother. 33:35-40. 20. Marca, G., M. Veronese, and D. Petrescu. 1973. Enhancement of the bacteriostatic and bactericidal activities of chloramphenicol and thiamphenicol by normal serum in vitro. Chemotherapy
ANTIMICROB. AGENTS CHEMOTHER.
18:91-98. 21. Mett, H., K. Gyr, 0. Zak, and K. Vosbeck. 1984. Duodenalpancreatic secretions enhance bactericidal activity of antimicrobial drugs. Antimicrob. Agents Chemother. 26:35-38. 22. Moellering, R. C., Jr., and G. M. Eliopoulos. 1984. Activity of cefotaxime against enterococci. Diagn. Microbiol. Infect. Dis.
2:85S-90S. 23. Nikaido, H., and M. Vaara. 1985. Molecular basis of bacterial outer membrane permeability. Microbiol. Rev. 49:1-32. 24. Perl, T. M., M. A. Pfaller, A. Houston, and R. P. Wenzel. 1990. Effect of serum on the in vitro activities of 11 broad-spectrum antibiotic. Antimicrob. Agents Chemother. 34:2234-2239. 25. Peterson, L. R., and D. N. Gerding. 1980. Influence of protein binding of antibiotics on serum pharmacokinetics and extravascular penetration: clinically useful concepts. Rev. Infect. Dis. 2:340-348. 26. Pruul, H., and P. J. McDonald. 1988. Damage to bacteria by antibiotics in vitro and its relevance to antimicrobial chemotherapy: a historical perspective. J. Antimicrob. Chemother. 21: 695-700. 27. Pruul, H., and B. L. Reynolds. 1972. Interaction of complement and polymyxin with gram-negative bacteria. Infect. Immun. 6:709-717. 28. Reimer, L. G., C. W. Stratton, and L. B. Reller. 1981. Minimum inhibitory and bactericidal concentrations of 44 antimicrobial agents against three standard control strains in broth with and without human serum. Antimicrob. Agents Chemother. 19: 1050-1055. 29. Retsema, J. A., A. Girard, W. Shelkley, M. Manousos, M. Bright, R. Borovoy, L. Brennan, and R. Mason. 1987. Spectrum and mode of action of azithromycin (CP-62,993), a new 15membered-ring macrolide with improved potency against gramnegative bacteria. Antimicrob. Agents Chemother. 31:19391947. 30. Retsema, J. A., A. E. Girard, D. Girard, and W. B. Milisen. 1990. Relationship of high tissue concentrations of azithromycin to bactericidal activity and efficacy in vivo. J. Antimicrob. Chemother. 25(Suppl. A):83-89. 31. Reynolds, B. L., and H. Pruul. 1971. Sensitization of complement-resistant smooth gram-negative bacterial strains. Infect. Immun. 3:365-372. 32. Stratton, C. W., and L. B. Reller. 1977. Serum dilution test for bactericidal activity. 1. Selection of a physiological diluent. J. Infect. Dis. 136:187-194. 33. Sullam, P. M., T. A. Drake, M. G. Tauber, C. J. Hackbarth, and M. A. Sande. 1985. Influence of the developmental state of valvular lesions on the antimicrobial activity of cefotaxime in experimental enterococcal infections. Antimicrob. Agents Chemother. 27:320-323. 34. Washington, J. A., and A. L. Barry. 1974. Dilution test procedures, p. 410417. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1992, p. 10-16
0066-4804/92/010010-07$02.00/0 Copyright C 1992, American Society for Microbiology
Potentiation of Antibacterial Activity of Azithromycin and Other Macrolides by Normal Human Serum HENDRIK PRUUL* AND PETER J. McDONALD Department of Microbiology and Infectious Diseases, Flinders Medical Centre, Bedford Park 5042, Australia Received 22 April 1991/Accepted 16 October 1991
The interaction of azithromycin with normal human serum was examined in relation to serum protein binding, MIC, and kinetics of killing of bacteria. While the binding of azithromycin to serum proteins is low (8.5% at a concentration of 0.01 mM in 95% serum), the presence of 40% serum during the MIC test decreased MICs by 26-fold for serum-resistant Escherichia coli and 15-fold for Staphylococcus aureus. Erythromycin had a similar but lesser effect, while roxithromycin was less active against S. aureus in the presence of serum. The rate of killing of E. coli and S. aureus by azithromycin was increased in the presence of serum. The enhancement of antibiotic activity by serum was pH independent, and heat inactivation and preabsorption with homologous bacteria failed to inhibit enhancement by serum. The macromolecular incorporation of [3H]thymidine by E. coli continuously exposed to 2 ,ug of azithromycin per ml (0.25x the MIC) and 40% serum was decreased by 80% at pH 7.8 and by 48% at pH 7.2, while azithromycin alone failed to inhibit incorporation. Inhibition of nucleic acid biosynthesis at pH 7.2 in the presence of serum was also detected with sub-MICs of erythromycin, norfloxacin, and gentamicin but not roxithromycin. A diffusible serum factor was shown to interact with azithromycin to inhibit the growth of E. coli in an agar diffusion assay to detect antibiotic-serum synergy. MATERIALS AND METHODS
It is well recognized that antibiotics which are highly bound to serum proteins have reduced antibacterial activity when they are tested for in vitro activity in the presence of serum proteins, since only free drug is available for antibacterial activity (6). On the other hand, in vitro synergy between antibiotics and serum components, including antibody and complement, has been reported (7, 20, 27) and may also be expressed in the responses of patients during antimicrobial chemotherapy. Standard inhibitory testing of antibiotics is carried out in the absence of host factors, including serum components. Few MIC studies have attempted to include host factors, although it is established that several variables, including serum proteins, phosphates, osmolarity, divalent cations, and pH, can affect the activity of certain antimicrobial agents (32). Azithromycin is a ring-expanded macrolide derivative with a spectrum of activity similar to that of erythromycin, but it has increased in vitro activity against some genera of gram-negative bacteria, including Haemophilus influenzae and Escherichia coli (1, 29). Peak levels of azithromycin in serum do not normally exceed 0.5 ,ug/ml (9). However, levels of azithromycin in tissue are greater than those in serum by a factor of approximately 10-fold (9, 10, 30). In addition, azithromycin has been reported to eradicate Salmonella enteritidis in a mouse model of tissue infection (10). On the basis of the elevated levels of azithromycin in tissue and its in vivo activity against gram-negative bacteria, azithromycin has potentially useful inhibitory activity against a wide range of pathogens, including members of the family Enterobacteriaceae. In this study we determined the serum protein binding of azithromycin, erythromycin, and roxithromycin and compared their inhibitory activities against bacteria in the presence of normal serum under controlled pH conditions. *
Antibiotics. The antibiotics except azithromycin used in this study were obtained from commercial sources; azithromycin was provided by Pfizer PTY, Ltd. Stock solutions were prepared as indicated by the manufacturers and were stored at -20°C. Prior to experiments, the antibiotic solutions were diluted in the appropriate experimental media and used immediately. Radiolabeled ['4C]azithromycin was obtained from Pfizer Inc., Groton, Conn., and [N-methyl'4C]erythromycin was obtained from DuPont, Boston, Mass. Protein binding. The extent of protein binding in pooled normal human serum and purified human serum proteins (Sigma Chemical Co., St. Louis, Mo.) was determined by the ultrafiltration method by using Amicon Centrifree filters with a molecular weight exclusion of 10,000 (Amicon Corp., Lexington, Mass.). Binding was determined in 95% serum and antibiotic concentrations of 0.01 mM. The concentrations of antibiotics in prefiltration solutions and filtrates were determined in triplicate by microbiological assay against Micrococcus luteus ATCC 9341 and Bacillus subtilis ATCC 6633 and by radioassay (Beckman liquid scintillation counter, model LS 3801) of total and filtered radiolabeled antibiotic. Control assays of antibiotics were carried out on unfiltered antibiotic solutions in phosphate-buffered saline. Binding of the antibiotics to the filtration membrane was determined after filtration of the antibiotic in phosphatebuffered saline, and the value was subtracted from that determined in the filtrate. Bacteria and MIC determinations. The E. coli strains used were serum-resistant isolates from infected clinical material. The Staphylococcus aureus strains were the Wood 46 and the ATCC 25923 strains and clinical isolates from infected clinical material. The strains were maintained on blood agar plates and subcultured every 3 weeks. Prior to the experiments, the bacteria were grown overnight in Trypticase soy
Corresponding author. 10
VOL. 36, 1992
INTERACTION OF MACROLIDES, SERUM, AND BACTERIA
broth (TSB; Oxoid, Basingstoke, United Kingdom), and the logarithmic phase of growth was obtained by serial dilution in fresh growth medium. MIC determinations were carried out in TSB or Mueller-Hinton broth, which were supplemented with the various serum and serum protein factors at a final concentration of 40%. Human serum albumin (Cohn fraction V) and globulins (Cohn fraction II, III; predominantly gamma and beta proteins) were obtained from Sigma Chemical Co. A modification of the tube dilution method described by Washington and Barry (34) was used. Briefly, immediately prior to bacterial inoculation, the medium was adjusted to pH 7.2, unless otherwise stated, sterilized by filtration, and dispensed in 0.4-ml portions in the MIC dilution tubes. Twofold dilutions of the antibiotics were carried out, and the tubes were inoculated with bacteria to yield 1 x 105 to 5 x 105 CFU/ml. The results were determined visually after 18 h of incubation at 37°C. Sera. Human serum was obtained from eight normal donors and was pooled. The freshly collected serum was divided into 1-ml portions, stored at -70°C, and thawed immediately before the experiment. Serum lacking complement activity was prepared by heating serum at 56°C for 30 min. Serum depleted of specific antibody was prepared by a modification of a previously described method (31). Briefly, freshly thawed serum was incubated with 1010 homologous bacteria for 30 min over ice. The absorption was carried out two times, and bacteria were removed by centrifugation and filtration. Determination of kinetics of killing. The experimental tubes containing 1.8 ml of pH-adjusted TSB with or without 40% serum were inoculated with 0.2 ml of 5 x 107 bacteria and incubated at 37°C for 4 h. The tubes were vigorously mixed (200 oscillations per min) throughout the incubation period. The pH was measured with a pH electrode with an impedance of less than 1,000 MQl (TPS Pty. Ltd., Brisbane, Australia), and the pH was maintained by the addition of 0.1 M NaOH or 0.1 M HCl at 45- to 60-min intervals. Not more than 12% (vol/vol) was added during the pH adjustments. Viable bacteria were determined by sampling 20 ,u1 into sterile saline solution. Portions were plated onto nutrient agar plates and incubated for 18 h at 37°C, and the CFU was counted. CFU was adjusted for the volumes added during
pH adjustment. Determination of [3H[thymidine incorporation. Bacteria incubated in 0.4 ml of pH-adjusted TSB with and without serum and antibiotic. After 100 min, 0.18 MBq (5% [vol/vol]) of [3H]thymidine (specific activity, 1.63 TBq/ mmol; Amersham International Ltd., Amersham, United Kingdom) per ml was added to the experimental tubes, the tubes were incubated for an additional 30 min, and triplicate 5-,ul samples were dispensed into 160 ,u1 of 8% trichloroacetic acid. Precipitated macromolecules were harvested on glass fiber discs by using a PHD cell harvester (Cambridge Technology Inc., Cambridge, Mass.), and the incorporated radioactivity was determined with a Beckman liquid scintillation counter. Determination of synergy between antibiotic and serum by diffusion in agar. Solid bacterial growth medium was prewere
pared by adding 1% (wt/vol) agar (Oxoid) to TSB, and the TSB-agar was liquefied at 121°C for 15 min and was allowed to cool to 55°C. The pH was adjusted to 7.4, and 5 x 106 E. coli per ml of TSB-agar was added. Then, 12.5 ml of the molten inoculated TSB-agar was added to 90-mm-diameter petri dishes, allowed to solidify on a level surface, and stored at 4°C. Wells (diameter, 6 mm) and troughs (30 by 2.5 mm) were cut into the agar, and 25 ,u1 of the test antibiotic was
11
TABLE 1. Protein binding of macrolides antibiotics in serum and effect of serum on MIC MIC (p.g/ml)b at pH:
Bacterium
Antibiotic
Serum binding?
7.2 No serum
E. coli
8 Serum
No
8.5 6.8 0.26 0.7 Azithromycin Erythromycin 39 80 14 13 54 Roxithromycin .95 .160 80
S. aureus Azithromycin Erythromycin Roxithromycin
Serum
serum
0.011 0.33 21
0.56 0.036 0.049 0.006 0.39 0.11 0.06 0.03 0.7 3.7 0.24 1.9
a Antibiotic (0.01 mM) was incubated with rotation for 1 h at 37°C with pooled 95% normal human serum. b Expressed as the geometric mean of at least seven independent determinations.
added to the wells. Diffusion of the antibiotic was allowed to occur for 2 h at 4°C prior to adding the test serum or control buffers (0.1 ml) to the troughs. The plates were incubated for 16 h at 37°C. Enhanced antibacterial activity was indicated by an increase in the zone of inhibition of growth between the antibiotic well and the trough. RESULTS MIC in the presence of serum. The mean MICs of the three macrolide antibiotics tested in TSB at initial pHs of 7.2 and 8 are presented in Table 1. The MICs for S. aureus ATCC 25923 and Wood 46 and the E. coli strain against azithromycin were markedly reduced by increasing the pH of the serum-free growth medium. MICs for S. aureus ATCC 25923 and Wood 46 did not differ, and the MICs for these two strains are combined in Tables 1 and 2. By increasing the pH from 7.2 to 8, the MIC for E. coli was reduced 9-fold and that for S. aureus was reduced 11-fold. Similar decreases in the MICs of erythromycin and roxithromycin occurred when the pH was increased to 8. Serum had a marked effect on the MIC of azithromycin at all pH levels tested. The MIC for E. coli, determined at pH 7.2, decreased from 6.8 to 0.26, a 26-fold reduction, and at pH 8 the decrease in the MIC was 60-fold. The decrease in the MIC of azithromycin for S. aureus in the presence of serum was 15-fold at pH 7.2 and 8-fold at pH 8. The corresponding changes in the MIC in Mueller-Hinton broth at pH 7.2 were 17-fold for E. coli and 40-fold for S. aureus (unpublished data). Serum also lowered the MICs for both E. coli and S. aureus when they were tested with erythromycin in both TSB and Mueller-Hinton broth. However, the MIC of roxithromycin against S. aureus was increased fivefold at pH 7.2 and eightfold at pH 8, while against E. coli, the MICs were decreased by approximately twofold only. Table 1 also includes data on the serum protein binding of azithromycin, erythromycin, and roxithromycin. Azithromycin binding was low and was confined mainly to alpha globulins (unpublished data). Binding of erythromycin was moderate, while .95% of roxithromycin was protein bound. Effect of serum depletion and purified serum proteins on MIC. The MICs of azithromycin and erythromycin were determined in TSB with 40% antibody-depleted serum and serum lacking complement activity, and the results were compared with those obtained with medium containing 40%
12
PRUUL AND McDONALD
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. Effect of serum depletion and purified serum proteins on the MICs of macrolide antibiotics MIC (>tg/ml)' Test medium
E. coli
S. aureus
AZI
ERY
AZI
ERY
TSB
6.8
80
0.56
0.39
40% Serum 40%o Abs-serumb 40% C-def serumc Albumin (70 mg/ml) Globulins (20 mg/ml)
0.26 0.4 0.34 7.5 6.7
6.5 12.5 12.1 .80 .80
0.026 0.038 0.04 0.53 0.67
0.11 0.16 0.14 0.24 0.34
a Expressed as the geometric mean of at least four independent determinations at an initial pH of 7.2. AZI, azithromycin; ERY, erythromycin. b Abs-serum, serum depleted of specified antibody. I C-def serum, serum lacking complement activity.
normal serum (Table 2). The effect of the addition of purified human serum proteins to TSB was also determined. No statistically significant changes (Student's t test) in serumdependent enhanced antibiotic activity against E. coli was detected when complement-inactivated serum or antibodydepleted serum was substituted for normal serum. The addition of 70 mg of albumin (Cohn fraction V) per ml and mixtures of beta and gamma globulins (Cohn fractions II and III) at 20 mg/ml failed to mimic the enhancing effect of serum. Similar results were obtained with S. aureus. pH-controlled kinetics of killing of bacteria by serum and sub-MICs of azithromycin. E. coli was incubated in TSB and 40% serum, and the pH was maintained at pH 7.2 throughout the time course of the experiment. The results are given in Fig. 1. In the presence of 5 ,ug of azithromycin per ml and
40% serum, there was greater than 2-log1o-unit killing at 90 min compared with a 1.25-log1o-unit killing in unsupplemented TSB. Azithromycin was bactericidal against E. coli at sub-MICs between 5 and 1 ,ug/ml only in the presence of serum. The pH of the medium did not vary by greater than 0.25 units during the course of the determinations of viable bacteria under controlled-pH conditions. The effects of various pHs on the bactericidal activity of sub-MICs of azithromycin were determined (Table 3). A variation of the pH of serum-TSB had no effect on the growth of E. coli in the absence of azithromycin, except at pH 7.8. At all concentrations of azithromycin in the absence of added serum, there was an increase in bactericidal activity with increasing pH. The addition of 40% serum enhanced the bactericidal activity of azithromycin at all pH levels tested except at elevated pH in the presence of 1 ,ug or greater of azithromycin per ml. Under these conditions, azithromycin alone had significant bactericidal activity. The rate of killing of S. aureus by azithromycin at pH 7.2 was slower than that of E. coli (Fig. 2). Even at lOx the MIC, there was less than 1-log1o-unit killing of S. aureus, compared with a 1.65-log1o-unit killing of E. coli within 2 h at 1 x the MIC. However, the effect of serum on the activity of azithromycin was similar. At lOx the MIC, azithromycin was bactericidal against S. aureus in both the presence and absence of serum and there was approximately a 0.7-log1ounit increased killing after 2 h of incubation in the presence of serum. At sub-MICs (.0.25 ,ug/ml), azithromycin retained antibacterial activity in the presence of serum, indicating a potentiation of azithromycin activity by serum. In the absence of serum, bacterial growth occurred at azithromycin concentrations of less than 0.5 ,ug/ml. Effect of serum on [3H]thymidine incorporation. The effect of sub-MICs of azithromycin on the ability of E. coli to
LL
0 0
0
0-
-1i ~ 0
I
1
I
iIIII
1 4 0 2 3 4 Time (hours) FIG. 1. Kinetics of killing of E. coli by azithromycin in the presence of 40% normal human serum at pH 7.2. Each datum point indicates the mean and 1 standard deviation. (A) 5 ,u.g of azithromycin per ml; (B) 2 ,ug of azithromycin per ml; (C) 1 ,ug of azithromycin per ml; (D) 0.5 ,g of azithromycin per ml. 0, 40% serum; 0, no serum; A, no antibiotic, 40% serum. 2
3
INTERACTION OF MACROLIDES, SERUM, AND BACTERIA
VOL. 36, 1992
13
TABLE 3. Effect of pH on the bactericidal activity of azithromycin against E. coli in the presence of 40% serum log1o change in CFU at 2 h at pH':
Medium
7.2
7.4
7.6
7.8
2 ,ug of AZIb per ml 2 p.g of AZI per ml + serum
0.13 (0.36)" -1.92 (0.3)
-1.26 (0.41) -2.07 (0.14)
-2.15 (0.46) -2.4 (0.23)
-2.5 (0.5) -1.75 (0.24)
1 p.g of AZI per ml 1 ,ug of AZI per ml + serum
1.11 (0.18) -0.82 (0.38)
0.03 (0.19) -1.14 (0.2)
-0.23 (0.46) -2.27 (0.45)
-1.64 (0.17) -1.4 (0.4)
0.5 ,ug of AZI per ml 0.5 ,ug of AZI per ml + serum
1.85 (0.4) 0.75 (0.19)
1 (0.25) 0.02 (0.3)
0.87 (0.09) 0.01 (0.29)
0.13 (0.15) -1.3 (0.2)
Serum alone
1.4 (0.18)
1.46 (0.25)
1.42 (0.41)
0.72 (0.14)
a Expressed as the geometric mean (standard error of the mean) of at least three independent determinations. b AZI, azithromycin.
incorporate [3H]thymidine into macromolecular nucleic acid is shown in Fig. 3. Bacteria were incubated with various concentrations of azithromycin in the presence and absence of serum for 100 min before the addition of [3H]thymidine, and cells were harvested at 130 min. Under the conditions of these experiments, sub-MICs of azithromycin in the absence of serum failed to inhibit incorporation. At all concentrations of antibiotic tested in the presence of serum, inhibition of incorporation of thymidine was greater at pH 7.8 than it was at pH 7.2. To achieve 50% inhibition at pH 7.8, a concentration of 0.8 ,ug of azithromycin per ml was required, while at pH 7.2, 2 ,ug of azithromycin per ml was necessary to achieve the same degree of inhibition. Several antibiotics were compared for their ability to inhibit thymidine incorporation in the presence of serum (Table 4). All tested antibiotics except roxithromycin inhib-
ited thymidine uptake at pH 7.2. Norfloxacin at 0.25 x the MIC was the most effective, while ampicillin and chloramphenicol were not as effective as the two active macrolides azithromycin and erythromycin. An increase in the pH to 7.8 increased inhibition by the macrolides and gentamicin, but not by norfloxacin and ampicillin. Detection of the potentiation of azithromycin activity by serum in an agar diffusion assay. Diffusion of azithromycin in TSB-agar inhibited bacterial growth in clearly defined circular zones of inhibition, as demonstrated in Fig. 4B. Similar inhibition zones were observed with phosphate-buffered saline at pH values of 7.2 to 8. The patterns of inhibition differed when the troughs were loaded with normal serum (Fig. 4A). Elliptical zones were observed, with bacterial growth inhibition extending to a greater distance from the center of antibiotic diffusion when compared with the inhi-
LL
I
C
()
0
4 3 4 0 12 Time (hours) FIG. 2. Kinetics of killing of S. aureus by azithromycin in the presence of 40% normal human serum at pH 7.2. (A) 5 ,ug of azithromycin per ml; (B) 0.5 ,ug of azithromycin per ml; (C) 0.25 ,ug of azithromycin per ml; (D) 0.1 ,ug of azithromycin per ml. *, 40% serum; 0, no serum; A, no antibiotic, 40% serum. 0
1
2
3
PRUUL AND McDONALD
14
ANTIMICROB. AGENTS CHEMOTHER.
E 2 CD .0
50-
1000
1
2 3 Azithromycin (pg/ml)
4
5
FIG. 3. Inhibition of [3H]thymidine incorporation by E. coli in the presence of serum and sub-MICs of azithromycin. 0, pH 7.8; 0, pH 7.2. Each point represents the mean ± standard deviation of at least three independent determinations. [3H]thymidine uptake is expressed as percent uptake in the absence of serum.
bition zones with troughs loaded with buffer. Antibiotic potentiation by serum was critically dependent on the distance of diffusion between serum and antibiotic, and at a concentration of 75 ,ug of antibiotic per ml at pH 7.2, enhanced antibacterial activity was observed at diffusion distances of between 3 and 5 mm. Potentiation between erythromycin and roxithromycin with serum was unable to be determined because these antibiotics failed to inhibit the growth of E. coli in agar at suitable concentrations of these antibiotics. DISCUSSION The low serum protein binding of azithromycin shown in Table 1 is consistent with the previously described concentration-dependent binding properties of the drug (9). At a molarity equivalent to that of azithromycin, erythromycin binding is approximately fivefold higher than that of azithromycin. At these comparatively low levels of protein binding, an increase in the MIC in the presence of serum proteins is TABLE 4. Effect of serum on [3H]thymidine uptake by E. coli in the presence of sub-MICs of antibiotics Antibiotic
X
Azithromycin Erythromycin Roxithromycin Ampicillin Norfloxacin Gentamicin Chloramphenicol a Expressed as
MICb
0.25 0.25 0.25 0.5 0.25 0.25 0.25
%
[3H]thymidine uptake"
pH 7.2
pH 7.8
52.5 (8)c 55.9 (18) >100 76.3 (17) 10.5 (8) 58.2 (5) 81.1 (14)
20.3 (4) 25.5 (3) >100 75.5 (15) 11.7 (3) 18 (10) 67.1 (9)
ihe percent of uptake by E.
coli in the absence of 40%
serum. b
Determined in the absence of serum at pH 7.2. Numbers in parentheses indicate the standard error of the means of at least three independent determinations. c
FIG. 4. Synergy between azithromycin and serum against E. coli in TSB-agar. (A) Normal serum (trough). Upper wells, 75 p.g of azithromycin per ml; lower wells, (left to right) 40, 50, 75, and 100 p,g of azithromycin per ml. (B) Phosphate-buffered saline; pH 8 (trough). Azithromycin was present at the concentrations described above for panel A.
not expected (25). In contrast, roxithromycin is highly bound by whole serum, and the presence of serum in the MIC test increased the MIC against S. aureus fivefold at pH 7.2. Increased MICs in serum-supplemented broth have been reported previously (16a) for roxithromycin against grampositive organisms and for other highly protein bound antibiotics (5, 16, 24, 32). However, roxithromycin decreases the MIC for E. coli by two- to fourfold in serum (Table 1). The reason for this is not clear, but is is probably not due to complement since heat-inactivated serum retains synergistic activity (Table 2). Perhaps the affinity of roxithromycin for binding sites of E. coli is greater than that for serum proteins, resulting in a concentration gradient that reverses the binding to serum proteins. The inhibitory effect of protein binding of antibiotics is normally detected in the MIC test by comparing the MICs in broth and serum. Anomalous enhancement activity of serum has been reported (13, 18, 19, 28, 33). Serum enhancement of antibiotic activity can be attributed to various factors, including the influence of serum on growth of the bacteria (2), serum alpha-2-globulin (22), changes in the pH of the test media (8), antibacterial peptides (4), or as yet unidentified synergistic host factors (18, 19, 21). Leggett and Craig (19) have shown that the increases in the MICs of ceftriaxone and other highly protein bound cephalosporins observed in the presence of albumin were significantly less than those predicted when they were determined in serum-containing medium and were not predicted from the protein-binding properties of the antibiotics. This serum enhancement of cephalosporin activity was limited to gram-negative species. However, the serum enhancement with macrolides de-
VOL. 36, 1992
INTERACTION OF MACROLIDES, SERUM, AND BACTERIA
scribed here occurs with staphylococci as well as E. coli. Other body fluids have been shown to enhance antibacterial activity. In a study determining the activity of antimicrobial drugs in duodenal-pancreatic secretions, Mett et al. (21) demonstrated enhanced bactericidal activity of clioquinol, chloramphenicol, and trimethoprim against E. coli and S. aureus, while other antibiotics, including colistin and tetracycline, failed to do so. This activity was not dependent on pH or iron and was heat stable. Macrolide antibacterial activity is sensitive to the pH of the test medium, and the data in Table 1 confirm the previous findings of Barry et al. (1), and in addition, the data demonstrate that. MICs are reduced in the presence of serum at elevated pHs as well as at physiological pH levels. In this study the pHs of the MIC tubes were controlled by adjusting the pH prior to incubation. However, the pHs of serum preparations are known to be unstable (3). In addition, bacterial metabolites may also influence the pH of the test medium. These factors make the continuous control of pH in MIC tubes difficult. In order to control pH more closely and to determine the early kinetics of antibacterial activity, bacteria were exposed to the combined activity of serum and azithromycin under conditions in which the pH was monitored and adjusted in the first 4 h of interactions. The experiments demonstrate that at constant pH, the presence of serum enhances the extent and early rate of killing of bacteria. The bactericidal activity of azithromycin against E. coli develops rapidly, while that against S. aureus is slower to develop (Fig. 1 and 2, respectively). This is in contrast to the findings of Retsema et al. (29) and may be due to differences in the pH of the medium and the effect of bacterial metabolites on pH. Figure 2 demonstrates that the growth of S. aureus at a controlled pH of 7.2 remains suppressed at 0.1 ,ug of azithromycin per ml (0.2 x MIC), and at levels near the MIC, the antibiotic is bactericidal in the presence of serum. The achievable in vivo level of azithromycin in serum is approximately 0.5 ,ug/ml (9), and significant antistaphylococcal activity at this concentration can be expected in the presence of serum. Inhibition of E. coli at pH 7.2 requires 10 times increased levels of azithromycin; these concentrations are achievable in various tissues (9, 10) but not in serum. The combination of serum and azithromycin has powerful antimetabolic activity. At sub-MICs of antibiotic, incorporation of thymidine into macromolecular nucleic acid is dramatically inhibited in the presence of serum (Fig. 3). The effect was enhanced at an elevated pH because of the intrinsic increased activity of azithromycin at high pH. At physiological pH, approximately one-third of the MIC of azithromycin was required to inhibit nucleic acid metabolism by 50%. These results indicate that extensive metabolic disruption occurs under conditions which increase the bactericidal activity of the antibiotic. In addition, exposure of bacteria to antibiotic has been shown to alter bacterial surface structures that are not directly related to the site of action of the antibiotic (14, 15, 17) and that modify subsequent interactions with host factors, including serum components and neutrophils (26). Several other antibiotics tested also showed synergistic antimetabolic activity in the presence of serum, including erythromycin, gentamicin, and ampicillin, but not roxithromycin (Table 4). Only the two active macrolides and gentamicin demonstrated increased inhibitory activity at elevated pH, and this may have been due to their increased antibacterial activity under those conditions. The inhibitory effect in the presence of erythromycin required 20 ,ug of the antibiotic per ml, a level that is
15
not achievable in vivo, while azithromycin was active at levels that are achievable in tissue and serum. The MICs of azithromycin for most E. coli strains, as determined in conventional broth media, are greater than those that are achievable in the blood, suggesting that it would be ineffective in the eradication of these bacteria. However, the eradication of S. enteritidis by azithromycin, but not by cefaclor, which was shown to have similar in vitro potency, has been demonstrated in a mouse tissue model of liver and spleen infection (10). The positive response was attributed to the high levels achieved in tissue and the long half-life in tissue. In addition, potentiation of antibiotic activity by serum and a rapid onset of bactericidal activity may also contribute to the efficacy of azithromycin against gram-negative bacteria. The serum factor(s) responsible for the potentiation of macrolide activity and its mode of action are not known. Changes in the bacterial growth phase and the composition of bacterial surfaces brought about by serum factors may modify bacterial membrane permeability, alter antibiotic accumulation kinetics, or enhance antibiotic binding to active sites. Wild-type gram-negative bacteria with intact outer cell wall structures exclude hydrophobic agents (23). Since macrolides are hydrophobic (11), gram-negative bacteria are normally resistant to this class of antibiotics. Disruption of the hydrophobic barrier of gram-negative bacteria by permeabilizers (12) has been shown to increase the susceptibilities of these bacteria to antibacterial agents that are normally excluded by the intact outer cell wall. For example, wildtype gram-negative bacteria that are relatively resistant to polymyxin are sensitized after chelation treatment of the bacteria with EDTA-Tris (27). The increased susceptibilities of rough, cell wall-deficient bacteria compared with those of smooth strains to erythromycin and other hydrophobic antibiotics have been reviewed (11). We detected early inhibition of nucleic acid biosynthesis in the presence of sub-MICs of some antibiotics and serum, indicating increased intracellular permeation and activity of azithromycin and other antibiotics in the presence of serum factors. We postulate that normal human serum interacts with a range of bacteria to alter their susceptibilities to azithromycin and other macrolide antibiotics with low protein binding characteristics. While high levels of serum protein binding interferes with serum synergy against staphylococci, it may be retained against E. coli. ACKNOWLEDGMENT This work was supported by Pfizer Central Research Division, Pfizer Inc. REFERENCES 1. Barry, A. L., R. N. Jones, and C. Thornsberry. 1988. In-vitro activities of azithromycin (CP 62993), clarithromycin (A-56268; TE-031), erythromycin, roxythromycin, and clindamycin. Antimicrob. Agents Chemother. 32:752-754. 2. Brown, R. W., and P. Williams. 1985. Influence of substrate limitation and growth phase on sensitivity to antimicrobial agents. J. Antimicrob. Chemother. 15(Suppl. A):7-14. 3. Bryan, C. S., S. R. Marney, Jr., R. H. Afford, and R. E. Bryant. 1975. Gram-negative bacillary endocarditis: interpretation of the serum bactericidal test. Am. J. Med. 58:209-215. 4. Carroll, S. F., and R. J. Martinez. 1981. Antibacterial peptide from normal rabbit serum. Biochemistry 20:5973-5981. 5. Craig, W. A., and C. M. Kunin. 1976. Significance of serum protein and tissue binding of antimicrobial agents. Annu. Rev. Med. 27:287-300. 6. Craig, W. A., and P. G. Welling. 1977. Protein binding of
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antimicrobials: clinical pharmacokinetics and therapeutic implications. Clin. Pharmacokinet. 2:252-268. 7. Dutcher, B. S., A. M. Reynard, M. E. Beck, and R. K. Cunningham. 1978. Potentiation of antibiotic activity by normal serum. Antimicrob. Agents Chemother. 13:820-826. 8. Eagle, H., M. Levy, and R. Fleischman. 1952. The effect of pH of the medium on the antibacterial action of penicillin, streptomycin, chloramphenicol, terramycin, and bacitracin. Antibiot. Chemother. 11:563-575. 9. Foulds, G., R. M. Shepard, and R. B. Johnson. 1990. The pharmacokinetics of azithromycin in human serum and tissues. J. Antimicrob. Chemother. 25(Suppl. A):73-82. 10. Girard, A. E., D. Girard, A. R. English, T. D. Gootz, C. R. Cimochowski, J. A. Faiella, S. L. Haskell, and J. A. Retsema. 1987. Pharmacokinetics and in vivo studies with azithromycin (CP-62,993), a new macrolide with an extended half-life and excellent tissue distribution. Antimicrob. Agents Chemother. 31:1948-1954. 11. Hancock, R. E. W., and A. Bell. 1988. Antibiotic uptake into gram-negative bacteria. Eur. J. Microbiol. Infect. Dis. 7:713-720. 12. Hancock, R. E. W., and P. G. W. Wong. 1984. Compounds which increase the permeability of the Pseudomonas aeruginosa outer membrane. Antimicrob. Agents Chemother. 26:48-52. 13. Hardy, D. J., R. N. Swanson, R. A. Rode, K. Marsh, N. L. Shipkowitz, and J. J. Clement. 1990. Enhancement of the in vitro and in vivo activities of clarithromycin against Haemophilus influenzae by 14-hydroxy-clarithromycin, its major metabolite in humans. Antimicrob. Agents Chemother. 34:1407-1413. 14. Hirashima, A., G. Childs, and M. Inouye. 1973. Differential inhibitory effects of antibiotics on the biosynthesis of envelope proteins of Escherichia coli. J. Mol. Biol. 79:373-389. 15. Iida, K., and M. Koike. 1974. Cell-wall alterations of gramnegative bacteria by aminoglycoside antibiotics. Antimicrob. Agents Chemother. 5:95-97. 16. Jones, R. N., and A. L. Barry. 1987. Antimicrobial activity of ceftriaxone, cefotaxime, desacetylcefotaxime, and cefotaximedesacetylcefotaxime in the presence of human serum. Antimicrob. Agents Chemother. 31:818-820. 16a.Jones, R. N., and A. L. Barry. 1987. Unpredictable influence of human serum on antimicrobial activity of erythromycin and three oxime ether macrolides. Eur. J. Clin. Microbiol. 6:81-82. 17. Klainer, A. S., and R. R. B. Russell. 1974. Effect of the inhibition of protein synthesis on the Escherichia coli cell wall envelope. Antimicrob. Agents Chemother. 6:216-224. 18. Lang, S. D. R., G. L. Cameron, and P. R. Mullins. 1981. Anomalous effect of serum on the antimicrobial activity of cefoperazone. Drugs 22(Suppl. 1):52-59. 19. Leggett, J. E., and W. A. Craig. 1989. Enhancing effect of serum ultrafiltrates on the activity of cephalosporins against gramnegative bacteria. Antimicrob. Agents Chemother. 33:35-40. 20. Marca, G., M. Veronese, and D. Petrescu. 1973. Enhancement of the bacteriostatic and bactericidal activities of chloramphenicol and thiamphenicol by normal serum in vitro. Chemotherapy
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18:91-98. 21. Mett, H., K. Gyr, 0. Zak, and K. Vosbeck. 1984. Duodenalpancreatic secretions enhance bactericidal activity of antimicrobial drugs. Antimicrob. Agents Chemother. 26:35-38. 22. Moellering, R. C., Jr., and G. M. Eliopoulos. 1984. Activity of cefotaxime against enterococci. Diagn. Microbiol. Infect. Dis.
2:85S-90S. 23. Nikaido, H., and M. Vaara. 1985. Molecular basis of bacterial outer membrane permeability. Microbiol. Rev. 49:1-32. 24. Perl, T. M., M. A. Pfaller, A. Houston, and R. P. Wenzel. 1990. Effect of serum on the in vitro activities of 11 broad-spectrum antibiotic. Antimicrob. Agents Chemother. 34:2234-2239. 25. Peterson, L. R., and D. N. Gerding. 1980. Influence of protein binding of antibiotics on serum pharmacokinetics and extravascular penetration: clinically useful concepts. Rev. Infect. Dis. 2:340-348. 26. Pruul, H., and P. J. McDonald. 1988. Damage to bacteria by antibiotics in vitro and its relevance to antimicrobial chemotherapy: a historical perspective. J. Antimicrob. Chemother. 21: 695-700. 27. Pruul, H., and B. L. Reynolds. 1972. Interaction of complement and polymyxin with gram-negative bacteria. Infect. Immun. 6:709-717. 28. Reimer, L. G., C. W. Stratton, and L. B. Reller. 1981. Minimum inhibitory and bactericidal concentrations of 44 antimicrobial agents against three standard control strains in broth with and without human serum. Antimicrob. Agents Chemother. 19: 1050-1055. 29. Retsema, J. A., A. Girard, W. Shelkley, M. Manousos, M. Bright, R. Borovoy, L. Brennan, and R. Mason. 1987. Spectrum and mode of action of azithromycin (CP-62,993), a new 15membered-ring macrolide with improved potency against gramnegative bacteria. Antimicrob. Agents Chemother. 31:19391947. 30. Retsema, J. A., A. E. Girard, D. Girard, and W. B. Milisen. 1990. Relationship of high tissue concentrations of azithromycin to bactericidal activity and efficacy in vivo. J. Antimicrob. Chemother. 25(Suppl. A):83-89. 31. Reynolds, B. L., and H. Pruul. 1971. Sensitization of complement-resistant smooth gram-negative bacterial strains. Infect. Immun. 3:365-372. 32. Stratton, C. W., and L. B. Reller. 1977. Serum dilution test for bactericidal activity. 1. Selection of a physiological diluent. J. Infect. Dis. 136:187-194. 33. Sullam, P. M., T. A. Drake, M. G. Tauber, C. J. Hackbarth, and M. A. Sande. 1985. Influence of the developmental state of valvular lesions on the antimicrobial activity of cefotaxime in experimental enterococcal infections. Antimicrob. Agents Chemother. 27:320-323. 34. Washington, J. A., and A. L. Barry. 1974. Dilution test procedures, p. 410417. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C.
